University of Goettingen Medical School

Göttingen, Germany

University of Goettingen Medical School

Göttingen, Germany
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Doeppner T.R.,University of Goettingen Medical School | El Aanbouri M.,University of Goettingen Medical School | Dietz G.P.H.,Lundbeck | Dietz G.P.H.,Research Center for the Molecular Physiology of the Brain | And 4 more authors.
Neurobiology of Disease | Year: 2010

Neural precursor cells (NPC) are an interesting tool in experimental stroke research, but their therapeutic potential is limited due to poor long-term survival. We therefore in vitro transduced subventricular zone-(SVZ)-derived NPC with the anti-apoptotic fusion protein TAT-Bcl-xL and analyzed NPC survival, differentiation, and post-stroke functional deficits after experimental ischemia in mice. Survival of TAT-Bcl-xL-transduced NPC, which were injected at day 7 post-stroke into the ischemic striatum, was significantly increased at 4weeks after stroke. Increased survival of NPC was associated with reduced infarct injury and decreased post-stroke functional deficits. Animals grafted with TAT-Bcl-xL-transduced NPC showed an increased number of immature cells expressing the neuronal marker doublecortin. Since mature neuronal differentiation of NPC was not observed, reduced post-stroke injury cannot be attributed to enhanced neuronal regeneration, but rather to indirect by-stander effects of grafted NPC. In line with this, NPC-mediated neuroprotection of cortical neurons in vitro was associated with increased secretion of growth factors. Thus, in vitro transduction of cultivated NPC with TAT-Bcl-xL results in enhanced resistance of transplanted NPC followed by long-term neuroprotection and ameliorated functional deficits after transient focal cerebral ischemia in mice. © 2010 Elsevier Inc.

Doeppner T.R.,University of Goettingen Medical School | Dietz G.P.H.,Lundbeck | Dietz G.P.H.,Research Center for the Molecular Physiology of the Brain | Weise J.,University of Jena Medical School | And 2 more authors.
Experimental Neurology | Year: 2010

Endogenous neurogenesis persists in the subgranular zone (SGZ) of the adult rodent brain. Cerebral ischemia stimulates endogenous neurogenesis involving proliferation, migration and differentiation of SGZ-derived neural precursor cells (NPC). However, the biological meaning of this phenomenon is limited by poor survival of NPC. In order to study the effects of an acute neuroprotective treatment on hippocampal endogenous neurogenesis after transient cerebral ischemia in mice, we applied a fusion protein consisting of the TAT domain of the HI virus with the anti-apoptotic Bcl-xL. Intravenous injection of TAT-Bcl-xL resulted in reduced hippocampal cell injury for up to 4weeks after stroke as assessed by TUNEL and NeuN staining. This was in line with a TAT-Bcl-xL-mediated reduced postischemic microglia activation. Analysis of endogenous hippocampal cell proliferation revealed an increased number of BrdU+ cells in the TAT-Bcl-xL group 4weeks after stroke compared to animals treated with saline and TAT-HA (negative control). Cell proliferation in non-ischemic sham operated animals was not affected by TAT-Bcl-xL. Twenty-eight days after stroke co-expression of BrdU+ cells with the immature neuronal marker doublecortin was significantly increased in TAT-Bcl-xL animals. Although TAT-Bcl-xL treatment also resulted in an increased number of BrdU+ cells expressing the mature neuronal marker NeuN, the total amount of these cells was low. These data show that TAT-Bcl-xL treatment yields both postischemic sustained hippocampal neuroprotection and increased survival of NPC rather than an induction of endogenous neurogenesis itself. © 2010 Elsevier Inc.

Rutherford M.A.,University of Goettingen Medical School | Chapochnikov N.M.,University of Goettingen Medical School | Chapochnikov N.M.,University of Gottingen | Chapochnikov N.M.,Max Planck Institute for Dynamics and Self-Organization | And 2 more authors.
Journal of Neuroscience | Year: 2012

Mammalian cochlear spiral ganglion neurons (SGNs) encode sound with microsecond precision. Spike triggering relies upon input from a single ribbon-type active zone of a presynaptic inner hair cell (IHC). Using patch-clamp recordings of rat SGN postsynaptic boutons innervating the modiolar face of IHCs from the cochlear apex, at room temperature, we studied how spike generation contributes to spike timing relative to synaptic input. SGNs were phasic, firing a single short-latency spike for sustained currents of sufficient onset slope. Almost every EPSP elicited a spike, but latency (300-1500μs) varied with EPSP size and kinetics. When current-clamp stimuli approximated the mean physiological EPSC (≈300 pA), several times larger than threshold current (rheobase, ≈50 μA), spikes were triggered rapidly (latency,≈500μs) and precisely (SD,<50μs). This demonstrated the significance of strong synaptic input. However, increasing EPSC size beyond the physiological mean resulted in less-potent reduction of latency and jitter. Differences in EPSC charge and SGN baseline potential influenced spike timing less as EPSC onset slope and peak amplitude increased. Moreover, the effect of baseline potential on relative threshold was small due to compensatory shift of absolute threshold potential. Experimental first-spike latencies in response to a broad range of stimuli were predicted by a two-compartment exponential integrate-and-fire model, with latency prediction error of<100μs. In conclusion, the close anatomical coupling between a strong synapse and spike generator along with the phasic firing property lock SGN spikes to IHC exocytosis timing to generate the auditory temporal code with high fidelity. © 2012 the authors.

Doeppner T.R.,University of Duisburg - Essen | Kaltwasser B.,University of Duisburg - Essen | Bahr M.,University of Goettingen Medical School | Hermann D.M.,University of Duisburg - Essen
Frontiers in Cellular Neuroscience | Year: 2014

Systemic transplantation of neural progenitor cells (NPCs) in rodents reduces functional impairment after cerebral ischemia. In light of upcoming stroke trials regarding safety and feasibility of NPC transplantation, experimental studies have to successfully analyze the extent of NPC-induced neurorestoration on the functional level. However, appropriate behavioral tests for analysis of post-stroke motor coordination deficits and cognitive impairment after NPC grafting are not fully established. We therefore exposed male C57BL6 mice to either 45 min (mild) or 90 min (severe) of cerebral ischemia, using the thread occlusion model followed by intravenous injection of PBS or NPCs 6 h post-stroke with an observation period of three months. Post-stroke motor coordination was assessed by means of the rota rod, tight rope, corner turn, inclined plane, grip strength, foot fault, adhesive removal, pole test and balance beam test, whereas cognitive impairment was analyzed using the water maze, the open field and the passive avoidance test. Significant motor coordination differences after both mild and severe cerebral ischemia in favor of NPC-treated mice were observed for each motor coordination test except for the inclined plane and the grip strength test, which only showed significant differences after severe cerebral ischemia. Cognitive impairment after mild cerebral ischemia was successfully assessed using the water maze test, the open field and the passive avoidance test. On the contrary, the water maze test was not suitable in the severe cerebral ischemia paradigm, as it too much depends on motor coordination capabilities of test mice. In terms of both reliability and cost-effectiveness considerations, we thus recommend the corner turn, foot fault, balance beam, and open field test, which do not depend on durations of cerebral ischemia. © 2014 Doeppner, Kaltwasser, Bähr and Hermann.

Doeppner T.R.,University of Duisburg - Essen | Doeppner T.R.,University of Goettingen Medical School | Bretschneider E.,University of Goettingen Medical School | Doehring M.,University of Goettingen Medical School | And 9 more authors.
Acta Neuropathologica | Year: 2011

Cerebral ischemia stimulates endogenous neurogenesis. However, the functional relevance of this phenomenon remains unclear because of poor survival and low neuronal differentiation rates of newborn cells. Therefore, further studies on mechanisms regulating neurogenesis under ischemic conditions are required, among which ephrin-ligands and ephrin-receptors (Eph) are an interesting target. Although Eph/ephrin proteins like ephrin-B3 are known to negatively regulate neurogenesis under physiological conditions, their role in cerebral ischemia is largely unknown. We therefore studied neurogenesis, brain injury and functional outcome in ephrin-B3 -/- (knockout) and ephrin-B3 +/+ (wild-type) mice submitted to cerebral ischemia. Induction of stroke resulted in enhanced cell proliferation and neuronal differentiation around the lesion site of ephrin-B3 -/- compared to ephrin-B3 +/+ mice. However, prominent post-ischemic neurogenesis in ephrin-B3 -/- mice was accompanied by significantly increased ischemic injury and motor coordination deficits that persisted up to 4 weeks. Ischemic injury in ephrin-B3 -/- mice was associated with a caspase-3-dependent activation of the signal transducer and activator of transcription 1 (STAT1). Whereas inhibition of caspase-3 had no effect on brain injury in ephrin-B3 +/+ animals, infarct size in ephrin-B3 -/- mice was strongly reduced, suggesting that aggravated brain injury in these animals might involve a caspase-3-dependent activation of STAT1. In conclusion, post-ischemic neurogenesis in ephrin-B3 -/- mice is strongly enhanced, but fails to contribute to functional recovery because of caspase-3-mediated aggravation of ischemic injury in these animals. Our results suggest that ephrin-B3 might be an interesting target for overcoming some of the limitations of further cell-based therapies in stroke. © 2011 Springer-Verlag.

Doeppner T.R.,University of Duisburg - Essen | Doeppner T.R.,University of Goettingen Medical School | Ewert T.A.S.,University of Hamburg | Tonges L.,University of Goettingen Medical School | And 13 more authors.
Stem Cells | Year: 2012

Novel therapeutic concepts against cerebral ischemia focus on cell-based therapies in order to overcome some of the side effects of thrombolytic therapy. However, cell-based therapies are hampered because of restricted understanding regarding optimal cell transplantation routes and due to low survival rates of grafted cells. We therefore transplanted adult green fluorescence protein positive neural precursor cells (NPCs) either intravenously (systemic) or intrastriatally (intracerebrally) 6 hours after stroke in mice. To enhance survival of NPCs, cells were in vitro protein-transduced with TAT-heat shock protein 70 (Hsp70) before transplantation followed by a systematic analysis of brain injury and underlying mechanisms depending on cell delivery routes. Transduction of NPCs with TAT-Hsp70 resulted in increased intracerebral numbers of grafted NPCs after intracerebral but not after systemic transplantation. Whereas systemic delivery of either native or transduced NPCs yielded sustained neuroprotection and induced neurological recovery, only TAT-Hsp70-transduced NPCs prevented secondary neuronal degeneration after intracerebral delivery that was associated with enhanced functional outcome. Furthermore, intracerebral transplantation of TAT-Hsp70-transduced NPCs enhanced postischemic neurogenesis and induced sustained high levels of brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor, and vascular endothelial growth factor in vivo. Neuroprotection after intracerebral cell delivery correlated with the amount of surviving NPCs. On the contrary, systemic delivery of NPCs mediated acute neuroprotection via stabilization of the blood-brain-barrier, concomitant with reduced activation of matrix metalloprotease 9 and decreased formation of reactive oxygen species. Our findings imply two different mechanisms of action of intracerebrally and systemically transplanted NPCs, indicating that systemic NPC delivery might be more feasible for translational stroke concepts, lacking a need of in vitro manipulation of NPCs to induce long-term neuroprotection. © AlphaMed Press.

Doeppner T.R.,University of Duisburg - Essen | Kaltwasser B.,University of Duisburg - Essen | Teli M.K.,University of Duisburg - Essen | Teli M.K.,National Institute of Technology Calicut | And 4 more authors.
Cell Death and Disease | Year: 2014

Intravenous transplantation of neural progenitor cells (NPCs) induces functional recovery after stroke, albeit grafted cells are not integrated into residing neural networks. However, a systematic analysis of intravenous NPC delivery at acute and post-acute time points and their long-term consequences does not exist. Male C57BL6 mice were exposed to cerebral ischemia, and NPCs were intravenously grafted on day 0, on day 1 or on day 28. Animals were allowed to survive for up to 84 days. Mice and tissues were used for immunohistochemical analysis, flow cytometry, ELISA and behavioral tests. Density of grafted NPCs within the ischemic hemisphere was increased when cells were transplanted on day 28 as compared with transplantation on days 0 or 1. Likewise, transplantation on day 28 yielded enhanced neuronal differentiation rates of grafted cells. Post-ischemic brain injury, however, was only reduced when NPCs were grafted at acute time points. On the contrary, reduced post-ischemic functional deficits due to NPC delivery were independent of transplantation paradigms. NPC-induced neuroprotection after acute cell delivery was due to stabilization of the blood-brain barrier (BBB), reduction in microglial activation and modulation of both peripheral and central immune responses. On the other hand, post-acute NPC transplantation stimulated post-ischemic regeneration via enhanced angioneurogenesis and increased axonal plasticity. Acute NPC delivery yields long-term neuroprotection via enhanced BBB integrity and modulation of post-ischemic immune responses, whereas post-acute NPC delivery increases post-ischemic angioneurogenesis and axonal plasticity. Post-ischemic functional recovery, however, is independent of NPC delivery timing, which offers a broad therapeutic time window for stroke treatment. © 2014 Macmillan Publishers Limited All rights reserved.

Doeppner T.R.,University of Duisburg - Essen | Doeppner T.R.,University of Goettingen Medical School | Kaltwasser B.,University of Duisburg - Essen | Kaltwasser B.,University of Goettingen Medical School | And 4 more authors.
Journal of Cerebral Blood Flow and Metabolism | Year: 2013

Heat-shock protein 70 (Hsp70) protects against cerebral ischemia, which is attributed to its chaperone activity. However, recent reports also describe pro-inflammatory actions of Hsp70 via activation of Toll-like receptors (TLR). Using membrane-permeable transactivator of transcription (TAT)-Hsp70, we analyzed TAT-Hsp70-induced neuroprotection and its underlying mechanism after cerebral ischemia in mice. Infusion of TAT-Hsp70 reduced infarct volume and enhanced blood-brain barrier integrity on day 3 poststroke, when given no later than 12 hours. The latter was associated with reduction of microglial activation, although upregulation of pro-inflammatory TLR-2/4 was observed both in verum and in control animals. Nevertheless, protein abundance and nuclear translocation of downstream nuclear factor kappa B (NF-κB) as well as proteasomal degradation of the NF-κB regulator Ikappa B alpha (IκB-α) were significantly reduced by TAT-Hsp70. TAT-Hsp70-induced neuroprotection and functional recovery were restricted to 4 weeks only. However, TAT-Hsp70 provided an appropriate extracellular milieu for delayed intravenous transplantation of adult neural precursor cells (NPCs). Thus, NPCs that were grafted 28 days poststroke induced long-term neuroprotection for at least 3 months, which was not due to integration of grafted cells but rather due to paracrine effects of transplanted NPCs. Conclusively, TAT-Hsp70 ameliorates postischemic inflammation via proteasome inhibition, thus providing an appropriate extracellular milieu for delayed NPC transplantation and culminating in long-term neuroprotection. © 2013 ISCBFM.

Ramachandran B.,European Neuroscience Institute ENI | Ahmed S.,European Neuroscience Institute ENI | Zafar N.,University of Goettingen Medical School | Dean C.,European Neuroscience Institute ENI
Hippocampus | Year: 2015

Ethanol inhibits memory encoding and the induction of long-term potentiation (LTP) in CA1 neurons of the hippocampus. Hippocampal LTP at Schaffer collateral synapses onto CA1 pyramidal neurons has been widely studied as a cellular model of learning and memory, but there is striking heterogeneity in the underlying molecular mechanisms in distinct regions and in response to distinct stimuli. Basal and apical dendrites differ in terms of innervation, input specificity, and molecular mechanisms of LTP induction and maintenance, and different stimuli determine distinct molecular pathways of potentiation. However, lamina or stimulus-dependent effects of ethanol on LTP have not been investigated. Here, we tested the effect of acute application of 60 mM ethanol on LTP induction in distinct dendritic compartments (apical versus basal) of CA1 neurons, and in response to distinct stimulation paradigms (single versus repeated, spaced high frequency stimulation). We found that ethanol completely blocks LTP in apical dendrites, whereas it reduces the magnitude of LTP in basal dendrites. Acute ethanol treatment for just 15 min altered pre- and post-synaptic protein expression. Interestingly, ethanol increases the neurosteroid allopregnanolone, which causes ethanol-dependent inhibition of LTP, more prominently in apical dendrites, where ethanol has greater effects on LTP. This suggests that ethanol has general effects on fundamental properties of synaptic plasticity, but the magnitude of its effect on LTP differs depending on hippocampal sub-region and stimulus strength. © 2014 Wiley Periodicals, Inc.

Duwe S.,Catholic University of Leuven | De Zitter E.,Catholic University of Leuven | Gielen V.,Catholic University of Leuven | Moeyaert B.,Catholic University of Leuven | And 7 more authors.
ACS Nano | Year: 2015

"Smart fluorophores", such as reversibly switchable fluorescent proteins, are crucial for advanced fluorescence imaging. However, only a limited number of such labels is available, and many display reduced biological performance compared to more classical variants. We present the development of robustly photoswitchable variants of enhanced green fluorescent protein (EGFP), named rsGreens, that display up to 30-fold higher fluorescence in E. coli colonies grown at 37 °C and more than 4-fold higher fluorescence when expressed in HEK293T cells compared to their ancestor protein rsEGFP. This enhancement is not due to an intrinsic increase in the fluorescence brightness of the probes, but rather due to enhanced expression levels that allow many more probe molecules to be functional at any given time. We developed rsGreens displaying a range of photoswitching kinetics and show how these can be used for multimodal diffraction-unlimited fluorescence imaging such as pcSOFI and RESOLFT, achieving a spatial resolution of ∼70 nm. By determining the first ever crystal structures of a negative reversibly switchable FP derived from Aequorea victoria in both the "on"- and "off"-conformation we were able to confirm the presence of a cis-trans isomerization and provide further insights into the mechanisms underlying the photochromism. Our work demonstrates that genetically encoded "smart fluorophores" can be readily optimized for biological performance and provides a practical strategy for developing maturation- and stability-enhanced photochromic fluorescent proteins. © 2015 American Chemical Society.

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